A novel in vitro tubular model to recapitulate features of distal airways: The bronchioid.

BACKGROUND: Airflow limitation is the hallmark of obstructive pulmonary diseases, with the distal airways representing a major site of obstruction. Although numerous in vitro models of bronchi already exist, there is currently no culture system for obstructive diseases that reproduces the architecture and function of small airways. Here, we aimed to engineer a model of distal airways to overcome the limitations of current culture systems. METHODS: We developed a so-called bronchioid model by encapsulating human bronchial adult stem cells derived from clinical samples in a tubular scaffold made of alginate gel. RESULTS: This template drives the spontaneous self-organisation of epithelial cells into a tubular structure. Fine control of the level of contraction is required to establish a model of the bronchiole, which has a physiologically relevant shape and size. 3D imaging, gene expression and single-cell RNA-seq analysis of bronchioids made of bronchial epithelial cells revealed tubular organisation, epithelial junction formation and differentiation into ciliated and goblet cells. Ciliary beating is observed, at a decreased frequency in bronchioids made of cells from COPD patients. The bronchioid can be infected by rhinovirus. An air-liquid interface is introduced that modulates gene expression. CONCLUSION: Here, we provide a proof of concept of a perfusable bronchioid with proper mucociliary and contractile functions. The key advantages of our approach, such as the air‒liquid interface, lumen accessibility, recapitulation of pathological features and possible assessment of clinically relevant endpoints, will make our pulmonary organoid-like model a powerful tool for preclinical studies.

Rhinovirus infection of bronchial epithelium induces specific bronchial smooth muscle cell migration of severe asthmatic patients.

BACKGROUND: Patients with severe asthma show an increase in both exacerbation frequency and bronchial smooth muscle (BSM) mass. Rhinovirus (RV) infection of the bronchial epithelium (BE) is the main trigger of asthma exacerbations. Histological analysis of biopsies shows that a close connection between BE and hypertrophic BSM is a criterion for severity of asthma. OBJECTIVE: We hypothesized that RV infection of BE specifically increases BSM-cell migration from patients with asthma. METHODS: Serum samples, biopsies, or BSM cells were obtained from 86 patients with severe asthma and 31 subjects without asthma. BE cells from subjects without asthma were cultured in an air-liquid interface and exposed to RV-16. Migration of BSM cells was assessed in response to BE supernatant using chemotaxis assays. Chemokine concentrations were analyzed by transcriptomics and ELISAs. Immunocytochemistry, western blotting, and flow cytometry were used to quantify CXCR3 isoform distribution. CXCR3 downstream signaling pathways were assessed by calcium imaging and western blots. RESULTS: BSM cells from patients with severe asthma specifically migrated toward RV-infected BE, whereas those from subjects without asthma did not. This specific migration is driven by BE C-X-C motif chemokine ligand 10, which was increased in vitro in response to RV infection as well as in vivo in serum from exacerbating patients with severe asthma. The mechanism is related to both decreased expression and activation of the CXCR3-B-specific isoform in BSM cells from those with severe asthma. CONCLUSIONS: We have demonstrated a novel mechanism of BSM remodeling in patients with severe asthma following RV exacerbation. This study highlights the C-X-C motif chemokine ligand 10/CXCR3-A axis as a potential therapeutic target in severe asthma.

Crucial role of fatty acid oxidation in asthmatic bronchial smooth muscle remodelling.

BACKGROUND: Bronchial smooth muscle (BSM) remodelling in asthma is related to an increased mitochondrial biogenesis and enhanced BSM cell proliferation in asthma. Since mitochondria produce the highest levels of cellular energy and fatty acid ß-oxidation is the most powerful way to produce ATP, we hypothesised that, in asthmatic BSM cells, energetic metabolism is shifted towards the ß-oxidation of fatty acids. OBJECTIVES: We aimed to characterise BSM cell metabolism in asthma both in vitro and ex vivo to identify a novel target for reducing BSM cell proliferation. METHODS: 21 asthmatic and 31 non-asthmatic patients were enrolled. We used metabolomic and proteomic approaches to study BSM cells. Oxidative stress, ATP synthesis, fatty acid endocytosis, metabolite production, metabolic capabilities, mitochondrial networks, cell proliferation and apoptosis were assessed on BSM cells. Fatty acid content was assessed in vivo using matrix-assisted laser desorption/ionisation spectrometry imaging. RESULTS: Asthmatic BSM cells were characterised by an increased rate of mitochondrial respiration with a stimulated ATP production and mitochondrial ß-oxidation. Fatty acid consumption was increased in asthmatic BSM both in vitro and ex vivo. Proteome remodelling of asthmatic BSM occurred via two canonical mitochondrial pathways. The levels of carnitine palmitoyl transferase (CPT)2 and low-density lipoprotein (LDL) receptor, which internalise fatty acids through mitochondrial and cell membranes, respectively, were both increased in asthmatic BSM cells. Blocking CPT2 or LDL receptor drastically and specifically reduced asthmatic BSM cell proliferation. CONCLUSION: This study demonstrates a metabolic switch towards mitochondrial ß-oxidation in asthmatic BSM and identifies fatty acid metabolism as a new key target to reduce BSM remodelling in asthma.

Fibrocyte accumulation in the airway walls of COPD patients.

The remodelling mechanism and cellular players causing persistent airflow limitation in COPD remain largely elusive. We have recently demonstrated that circulating fibrocytes, a rare population of fibroblast-like cells produced by the bone marrow stroma, are increased in COPD patients during an exacerbation. We aimed to quantify fibrocyte density in situ in bronchial specimens from both control subjects and COPD patients, to define associations with relevant clinical, functional and computed tomography (CT) parameters, and to investigate the effect of the epithelial microenvironment on fibrocyte survival in vitro ("Fibrochir" study).A total of 17 COPD patients and 25 control subjects, all requiring thoracic surgery, were recruited. Using co-immunostaining and image analysis, we identified CD45+ FSP1+ cells as tissue fibrocytes, and quantified their density in distal and proximal bronchial specimens. Fibrocytes, cultured from the blood samples of six COPD patients, were exposed to primary bronchial epithelial cell secretions from control subjects or COPD patients.We demonstrate that fibrocytes are increased in both distal and proximal tissue specimens of COPD patients. The density of fibrocytes is negatively correlated with lung function parameters and positively correlated with bronchial wall thickness as assessed by CT scan. A high density of distal bronchial fibrocytes predicts the presence of COPD with a sensitivity of 83% and a specificity of 70%. Exposure of fibrocytes to COPD epithelial cell supernatant favours cell survival.Our results thus demonstrate an increased density of fibrocytes within the bronchi of COPD patients, which may be promoted by epithelial-derived survival-mediating factors.

Impact of donor, recipient and matching on survival after high emergency lung transplantation in France.

INTRODUCTION: Since July 2007, the French high emergency lung transplantation (HELT) allocation procedure prioritises available lung grafts to waiting patients with imminent risk of death. The relative impacts of donor, recipient and matching on the outcome following HELT remain unknown. We aimed at deciphering the relative impacts of donor, recipient and matching on the outcome following HELT in an exhaustive administrative database. METHODS: All lung transplantations performed in France were prospectively registered in an administrative database. We retrospectively reviewed the procedures performed between July 2007 and December 2015, and analysed the impact of donor, recipient and matching on overall survival after the HELT procedure by fitting marginal Cox models. RESULTS: During the study period, 2335 patients underwent lung transplantation in 11 French centres. After exclusion of patients with chronic obstructive pulmonary disease/emphysema, 1544 patients were included: 503 HELT and 1041 standard lung transplantation allocations. HELT was associated with a hazard ratio for death of 1.41 (95% CI 1.22-1.64; p<0.0001) in univariate analysis, decreasing to 1.32 (95% CI 1.10-1.60) after inclusion of recipient characteristics in a multivariate model. A donor score computed to predict long-term survival was significantly different between the HELT and standard lung transplantation groups (p=0.014). However, the addition of donor characteristics to recipient characteristics in the multivariate model did not change the hazard ratio associated with HELT. CONCLUSIONS: This exhaustive French national study suggests that HELT is associated with an adverse outcome compared with regular allocation. This adverse outcome is mainly related to the severity status of the recipients rather than donor or matching characteristics.

Bronchial Smooth Muscle Remodeling in Nonsevere Asthma.

RATIONALE: Increased bronchial smooth muscle (BSM) mass is a key feature of airway remodeling that classically distinguishes severe from nonsevere asthma. Proliferation of BSM cells involves a specific mitochondria-dependent pathway in individuals with severe asthma. However, BSM remodeling and mitochondrial biogenesis have not been examined in nonsevere asthma. OBJECTIVES: We aimed to assess whether an increase in BSM mass was also implicated in nonsevere asthma and its relationship with mitochondria and clinical outcomes. METHODS: We enrolled 34 never-smoker subjects with nonsevere asthma. In addition, we recruited 56 subjects with nonsevere asthma and 19 subjects with severe asthma as comparative groups (COBRA cohort [Cohorte Obstruction Bronchique et Asthme; Bronchial Obstruction and Asthma Cohort; sponsored by the French National Institute of Health and Medical Research, INSERM]). A phenotypic characterization was performed using questionnaires, atopy and pulmonary function testing, exhaled nitric oxide measurement, and blood collection. Bronchial biopsy specimens were processed for immunohistochemistry and electron microscopy analysis. After BSM remodeling assessment, subjects were monitored over a 12-month period. MEASUREMENTS AND MAIN RESULTS: We identified characteristic features of remodeling (BSM area >26.6%) and increased mitochondrial number within BSM in a subgroup of subjects with nonsevere asthma. The number of BSM mitochondria was positively correlated with BSM area (r = 0.78; P < 0.001). Follow-up analysis showed that subjects with asthma with high BSM had worse asthma control and a higher rate of exacerbations per year compared with subjects with low BSM. CONCLUSIONS: This study reveals that BSM remodeling and mitochondrial biogenesis may play a critical role in the natural history of nonsevere asthma (Mitasthme study). Clinical trial registered with www.clinicaltrials.gov (NCT00808730).

Blood fibrocytes are recruited during acute exacerbations of chronic obstructive pulmonary disease through a CXCR4-dependent pathway.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by peribronchial fibrosis. The chronic course of COPD is worsened by recurrent acute exacerbations. OBJECTIVE: The aim of the study was to evaluate the recruitment of blood fibrocytes in patients with COPD during exacerbations and, subsequently, to identify potential mechanisms implicated in such recruitment. METHODS: Using flow cytometry, we quantified circulating fibrocytes and characterized their chemokine receptor expression in 54 patients with COPD examined during an acute exacerbation (V1) and 2 months afterward (V2) and in 40 control subjects. The role of the chemokines CXCL12 and CCL11 in fibrocyte migration was investigated by using a chemotaxis assay. Patients were followed for up to 3 years after V1. RESULTS: We demonstrated a significantly increased number of circulating fibrocytes at V1 compared with control subjects. The number of circulating fibrocytes decreased at V2. A high percentage of circulating fibrocytes during exacerbation was associated with increased risk of death. The percentage of fibrocytes at V2 was negatively correlated with FEV1, forced vital capacity, FEV1/forced vital capacity ratio, transfer lung capacity of carbon monoxide, and Pao2. Fibrocytes highly expressed CXCR4 and CCR3, the chemokine receptors for CXCL12 and CCL11, respectively. Fibrocytes collected from patients with COPD at V1 had increased chemotactic migration in response to CXCL12 but not to CCL11 compared with those from control subjects. Plerixafor, a CXCR4 antagonist, decreased fibrocyte migration to plasma from patients with exacerbating COPD. CONCLUSION: Blood fibrocytes are recruited during COPD exacerbations and related to mortality and low lung function. The CXCL12/CXCR4 axis is involved in such fibrocyte recruitment (Firebrob study; ClinicalTrials NCT01196832).

Selective dysfunction of p53 for mitochondrial biogenesis induces cellular proliferation in bronchial smooth muscle from asthmatic patients.

BACKGROUND: Increase of bronchial smooth muscle (BSM) mass is a crucial feature of asthma remodeling. The mechanisms of such an increased BSM mass are complex but involve enhanced mitochondrial biogenesis, leading to increased proliferation of BSM cells in asthmatic patients. The major tumor suppressor protein p53 is a key cell regulator involved in cell proliferation and has also been implicated in mitochondrial biogenesis. However, the role of p53 in BSM cell proliferation and mitochondrial biogenesis has not been investigated thus far. OBJECTIVE: We sought to evaluate the role of p53 in proliferation of BSM cells in asthmatic patients and mitochondrial biogenesis. METHODS: The expression of p53 was assessed both in vitro by using flow cytometry and Western blotting and ex vivo by using RT-PCR after laser microdissection. The role of p53 was assessed with small hairpin RNA lentivirus in both asthmatic patients and control subjects with BSM cell proliferation by using 5-bromo-2'-deoxyuridine and cell counting and in the expression of p21, BCL2-associated X protein, mitochondrial transcription factor A (TFAM), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α). RESULTS: Twenty-nine patients with moderate-to-severe asthma and 26 control subjects were enrolled in the study. p53 expression was increased in BSM from asthmatic patients both ex vivo and in vitro, with a decreased interaction with mouse double minute 2 homolog (Mdm2) and an increased phosphorylation of serine 20. p53 did not inhibit the transcription of both TFAM and PGC-1α in BSM cells from asthmatic patients. As a consequence, p53 is unable to slow the increased mitochondrial biogenesis and hence the subsequent increased proliferation of BSM cells in asthmatic patients. CONCLUSION: This study suggests that p53 might act as a new potential therapeutic target against BSM remodeling in asthmatic patients.